The present invention relates to diagnostic methods for detection and quantification of lipoproteins and cholesterol associated with lipoproteins. More particularly, the invention relates to assay methods for direct measurement of apolipoprotein B (apoB) associated very low density lipoproteins (VLDL) and cholesterol associated with VLDL using specific monoclonal antibodies.
Very low density lipoprotein (VLDL) constitutes one of the major plasma lipoproteins. VLDL particles are synthesized in the liver and are involved in triglyceride metabolism and transport of these lipids from the liver. The end products of VLDL catabolism are low density lipoproteins (LDL), another major class of lipoprotein particles in plasma.
It has been suggested that disturbances in the metabolism of apoB containing lipoproteins such as VLDL and LDL correlate with incidences of atherosclerosis (Hurt-Camejo et al. (1997) Arteriosclerosis, Thrombosis, and Vascular Biology 17(6): 1011-1017). Furthermore, an increase in VLDL levels has been associated with hypertriglycerimedia, hyperlipidemia or familial combined hyperlipidemia (Betteridge (1989) Diabet Med 6: 195-218; Schaefer et al. (1985) New Engl J. Med 312: 1300-1310; Shaefer et al. (1993) Curr Opin Lipidol 4: 288-298). Hyperglyceridemia also has been shown to correlate with an increased incidence of coronary heart disease (Gianturco et al. (1991) Curr Opin Lipidol 2: 324-328; Manninen et al. (1992) Circulation 85: 37-45; Grundy et al. (1992) Arch Intern Med 152: 28-34). Many patients with hyperglycerimedia have very low levels of high density lipoprotein (HDL), another major lipoprotein in plasma. It is recommended to treat patients with coronary heart disease who concurrently have hypertriglyceridemia and low levels of HDL with drugs and pharmacologic reagents, even when these patients have acceptable levels of total and LDL cholesterol (Larsen et al. (1993) Curr Opin Lipidol 4: 34-40); Stein and Myers (1995) Clin Chem 41: 1421-1426).
Two methods are presently used for the quantitation of VLDL, both of which involve the measurement of VLDL-cholesterol. The first method uses the factor triglyceride/5 as VLDL-cholesterol concentration (Friedewald et al. (1972) Clin Chem 18: 499-502). In this method, it is assumed that all plasma triglycerides are associated with VLDL and chylomicrons and that other VLDL remnants are not present. Chylomicrons are microscopic lipid particles that appear in the blood transiently after a fat-containing meal, are rich in triglycerides and usually have no significant effect on the total-cholesterol concentration. Although these assumptions are not strictly true, the factor triglyceride/5 usually provides good measure of VLDL-cholesterol when the subject is fasting and the triglyceride concentrations do not exceed 400 mg/dL.
The second method for quantitating VLDL uses ultracentrifugation. In this method, an aliquot of plasma is used to measure the total cholesterol concentration in the sample. A second aliquot of plasma is centrifuged (105,000xc3x97 g) at a plasma density concentration of 1.006 g/mL for 18 hours at 4xc2x0 C. After centrifugation, the upper layer containing VLDL is quantitatively removed and the cholesterol concentration in the isolated VLDL is measured. Alternatively, an aliquot of the remaining bottom layer, which does not contain VLDL, is used to measure the cholesterol concentration ([d  greater than 1.006 g/mL chol]). The cholesterol concentration of VLDL ([VLDL-chol]) is then calculated using the following equation:
[VLDL-chol]=[Total-chol]xe2x88x92[d greater than 1.006 g/mL chol]
Both methodologies suffer from a variety of problems. For example, the use of the factor triglyceride/5 is unacceptable in cases where a subject is not fasting, or where triglyceride concentrations exceed 400 mg/dL. Moreover, this method should not be used for Type III hyperlipoproteinemic patients that contain floating beta-VLDL (Belcher et al. (1991) Methods for Clinical Laboratory Measurement of Lipid and Lipoprotein Risk Factors, Eds. Rifai and Warnick, MCC Press, Washington, D.C., pp. 75-86). Although several studies have been conducted to determine better ways to measure VLDL-cholesterol concentrations (McNamara et al. (1990) Clin Chem 36: 36-42; Warnick et al. (1990) Clin Chem36: 15-19; Delong et al. (1986) JAMA 256: 2372-2377), no significant improvement has yet been made.
The problem with the ultracentrifugation method of VLDL-cholesterol quantitation is that it is both time consuming and expensive to perform. Furthermore, since the method requires specialized equipment, facilities and laboratory skills, it is not suitable for routine analysis of patient samples. To complicate these matters, no alternative methodologies for measuring VLDL, such as assays which measure apoB associated with VLDL, are currently available, either for analysis of patient samples or for research purposes. Thus, there is a need for rapid, easily performed, accurate and cost effective methods for quantitating VLDL.
An objective of this invention is to generate monoclonal antibodies that are specific for VLDL. Another object of this invention is to develop a method of directly measuring apolipoprotein B-100 (apoB) and cholesterol associated with VLDL from plasma easily, cheaply, quickly and accurately without the need of highly trained technicians or expensive equipment such as ultracentrifuges. Yet another object of this invention is to directly measure VLDL-cholesterol without the analytical variability generally associated with the present method of quantitative removal of VLDL layer in the ultracentrifugation method even with highly trained technicians.
In one embodiment, the present invention provides a method for determining the amount of apoB associated with VLDL in a sample comprising the steps of: (a) mixing a sample and a VLDL-specific binding agent for a time and under conditions to form binding-agent-VLDL complexes; and (b) determining the amount of apoB associated with VLDL bound to the binding-agent-VLDL complexes. In a preferred embodiment, the VLDL-specific binding agent is coupled to a solid support. In a more preferred embodiment, the solid support is separated from the sample before determining the amount of apoB bound to the binding-agent-VLDL complexes. Preferred solid supports include nitrocellulose, latex, nylon, polystyrene beads, particles, magnetic particles, and glass fiber. In these embodiments, the VLDL-specific binding agent is an antibody or fragment thereof that binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding. Preferably, the antibody is a monoclonal antibody. More preferably, the monoclonal antibody is selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211. A most preferred monoclonal antibody is 18-358-211.
In yet another embodiment, the method further comprises the step of separating the binding-agent-VLDL complexes prior to determining the amount of apoB associated with VLDL. In a preferred method of this embodiment, the VLDL-specific binding agent is conjugated to a first charged substance and the separation step comprises contacting the binding-agent-VLDL complexes with an insoluble solid phase material which is oppositely charged with respect to the first charged substance, such that the solid phase material attracts and attaches to the first charged substance and separating the solid phase material and the sample. Preferably, the charged substances are anionic and cationic monomers or polymers.
In another embodiment, the invention provides a method for determining the amount of apoB associated with VLDL in a sample comprising the steps of: contacting the sample with an indicator reagent wherein the indicator reagent is a monoclonal antibody or fragment thereof that specifically binds to apoB associated with VLDL and with a solid support coated with VLDL for a time and under conditions to permit binding of the indicator reagent with the VLDL in the sample and with the bound VLDL and determining the amount of apoB associated with VLDL in the test sample by detecting the reduction in binding of the indicator reagent to the solid support as compared to the signal generated from a negative sample to indicate the presence of VLDL in the test sample. In this embodiment, the indicator reagent is preferably the monoclonal antibody 18-358-211.
In another embodiment, the invention provides a method for determining the amount of cholesterol associated with VLDL in a sample and comprises the steps of mixing a sample and a VLDL-specific binding agent for a time and under conditions to form binding-agent-VLDL complexes; and determining the amount of cholesterol bound to the binding-agent-VLDL complexes. In a preferred embodiment, the VLDL-specific binding agent is coupled to a solid support. In a more preferred embodiment, the solid support is separated from the sample before determining the amount of cholesterol bound to the binding-agent-VLDL complexes. Preferred solid supports include nitrocellulose, latex, nylon, polystyrene, beads, particles, magnetic particles, and glass fiber. In these embodiments, the VLDL-specific binding agent is an antibody or fragment thereof that binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding. Preferably, the antibody is a monoclonal antibody. More preferably, the monoclonal antibody is selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211.
In one alternative embodiment, determining the amount of cholesterol comprises releasing the cholesterol bound to the binding agent-VLDL complexes and measuring the amount of cholesterol released. In a second alternative embodiment, determining the amount of cholesterol comprises mixing the binding-agent-VLDL complexes with a cholesterol specific binding agent coupled to a detectable label for a time and under conditions suitable to form binding-agent-VLDL-cholesterol specific binding agent complexes and determining the amount of label bound to the binding-agent-VLDL-cholesterol specific binding agent complex.
In yet another embodiment, the method further comprises the step of separating the binding-agent-VLDL complexes prior to determining the amount of cholesterol associated with VLDL. In a preferred embodiment, the VLDL-specific binding agent is conjugated to a first charged substance and the separation step comprises contacting the binding-agent-VLDL complexes with an insoluble solid phase material which is oppositely charged with respect to the first charged substance, such that the solid phase material attracts and attaches to the first charged substance and separating the solid phase material and the sample. Preferably, the charged substances are anionic and cationic monomers or polymers. One alternative embodiment of this method involves releasing the cholesterol bound to the binding-agent-VLDL complexes and measuring the amount of cholesterol released. A second alternative embodiment involves mixing the binding-agent-VLDL complexes with a cholesterol specific binding agent coupled to a detectable label such that a second complex is formed and determining the amount of label bound to the second complex.
The present invention also provides an antibody or fragment thereof specific for VLDL wherein the antibody binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding. Preferably, the antibody is selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211. A most preferred antibody is produced by a hybridoma cell line having ATCC Accession No. HB-12392.
The present invention also provides a hybridoma cell line that produces a monoclonal antibody which binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of VLDL binding. Preferably, the hybridoma cell line produces a monoclonal antibody selected from the group consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211. A most preferred hybridoma cell has ATCC Accession No. HB-12392.
The present invention yet further provides a monoclonal antibody specific for VLDL prepared by the method comprising the steps of: (a) immunizing a mouse or a rat with Apo CIII; (b) making a suspension of the mouse or rat spleen cells; (c) fusing the spleen cells with mouse or rat myeloma cells in the presence of a fusion promoter; (d) culturing the fused cells; (e) determining the presence of anti-VLDL antibody in the culture media; (f) cloning a hybridoma producing antibody that binds to substantially all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less than about 10% of VLDL binding, and to HDL at less than about 10% of LDL binding; and (g) obtaining the antibody from the hybridoma.